The Functions And Mechanisms Study Of C18-ceramides In Glioma

Objective: The major glycosphingolipids expression profiles in the human brain tumor tissues will be analyzed.We will also compare the differences between tumor tissues and controls,and will be looking for characteristic glycosphingolipids associated with tumor.The study aims to elucidate the important roles of glycosphingolipids in brain tumors.The profiles of ceramides in glioma and controls will be studied.We will also compare the differences between glioma and controls,and look for characteristic ceramide associated with glioma.The functions of characteristic ceramide in glioma will be studied.The potential signaling pathways that might induce the cell viability reduction and cell death via overexpression of CERS1 will be studied.We will also discuss the potential mechanism of C18-ceramide in glioma.Methods:(1)Glycosphingolipids were extracted by sonication with organic solvents from brain tumor samples and controls;(2)A modification of the method presented by Ciucanu and Costello was employed for per-N,O-methylation of glycosphingolipids;(3)Using mass spectrometry to characterize the structure of major glycosphingolipids from brain tumor samples;(4)The relative quantitative of obtained glycosphingolipids from samples were performed with mass spectrometry.The differences were compared between brain tumors and controls;(5)Ceramides were extracted by sonication with organic solvents from glioma tumor tissues and controls;(6)A modification was employed for per-N,O-methylation of ceramides;(7)Using mass spectrometry to characterize the structure of major ceramides from glioma and controls;(8)The relative quantitative of obtained ceramides from samples were performed with mass spectrometry.The differences were compared between glioma and controls;(9)CERS1 was overexpressed in glioma cells to increase the content of C18-ceramides;(10)The effects on cell cycle,cell viability and cell death were detected in glioma cell with CERS1 overexpression or exogenous C18-ceramide;(11)The effects on chemosensitivity to VM-26 were detected in glioma cell with CERS1 overexpression or exogenous C18-ceramide;(12)The gene expression microarray was employed to analyze the gene expression changes in glioma cells overexpressed CERS1;(13)The qRT-PCR and Western blot were employed to verify the results of microarray,and detected the effect on ER stress in glioma cells overexpressed CERS1.The CHOP RNAi was performed to verify the ER stress involvement;(14)The Western blot and confocal microscope were employed to detect the autophagy in glioma cells overexpressed CERS1,and the inhibitor of autophagy was used to verify the autophagy involvement;(15)The Western blot was employed to detect the effect on PI3K/AKT pathway in glioma cells overexpressed CERS1,and the activator of PI3K/AKT pathway was used to verify the PI3K/AKT involvement.Results:(1)Various glycosphingolipids in the brain tumors and controls were qualitatively analyzed used electrospray ionization tandem mass spectrometry,containing major neutral glycosphingolipids: GlcCer,LacCer,Gb3 and Gb4,major gangliosides: GM3,GM2,GM1,GD3,GD2,GD1,GT3,O-Ac-GM3,O-Ac-GD3 and O-Ac-GT3;(2)Relative quantitative of glycosphingolipids in the samples was analyzed.The results showed the contents of GlcCer were decreased in meningioma,hypophysoma and glioma compared with controls,and the contents of LacCer,Gb3 and Gb4 were increased in meningeoma,hypophysoma and glioma compared with controls;(3)We found the contents of C18-GSLs were higher in glioma than in controls;(4)The major gangliosides were GM1(70.36%)in controls,but GM3 were the major gangliosides in meningioma(39.94%),hypophysoma(41.30%)and glioma(37.36%);(5)C18-ceramide expression was higher in controls(mean=0.224)than in glioma patients(mean=0.106)(p < 0.001);(6)We improved the expression of CERS1 by pcDNA3.1(+)/CERS1 transfection,which exclusively synthesized C18-ceramide,in glioma cells U251 and A172;(7)CERS1 and C18-ceramide could decrease cell viability and increase cell death in glioma cells U251 and A172;(8)Overexpression of CERS1 or exogenous of C18-ceramide increased the sensitivity of U251 and A172 glioma cells to VM-26;(9)The gene expression microarray results showed that DDIT4(a negative regulator of mTOR),PIK3 CA,MAP1LC3β,eIF2α,ATF-6,ATF-3,CHOP,XBP-1 and ATF-4 had significant changes in U251 cells overexpressed CERS1 compared with control;(10)ER stress was activated by overexpression of CERS1 in U251 and A172 glioma cells.Loss function of CHOP(using CHOP siRNA)could recover the depressed cell viability caused by overexpression of CERS1,which could validate the activation of ER stress induced by overexpression of CERS1;(11)As a result,the conversion of LC3B-I to LC3B-II and reduction of p62 reflected the occurrence of autophagy in U251 and A172 glioma cells which CERS1 was overexpressed.CERS1 also significantly increased the punctate distribution and density of GFP-LC3 in U251 and A172 cells.The cell viability was increased in CERS1+3-MA group compared with CERS1 group in U251(P < 0.05)and A172(P < 0.05)cells,suggesting that 3-MA blocked the inhibitory effect of CERS1 on the cell viability.The results also indicated that CERS1 could induce lethal autophagy in U251 and A172 cells;(12)The observations suggested that the suppression of PI3K/AKT pathway activity contributed to CERS1 decreasing cell viability,and IGF-1 could block the effect of CERS1 on the reduction of cell viability;(13)The cell death induced by overexpression of CERS1 was independent of Bax and Bcl-2 in glioma cells.Conclusion: The glycosphingolipids contents in meningioma,hypophysoma and glioma were different from that of controls,and the contents of C18-GSLs were higher in glioma than in controls.C18-ceramide expression was decreased in glioma compared with controls,and overexpression of CERS1 or exogenous of C18-ceramide could decrease the cell viability,promote cell death and increase the sensitivity to VM-26 in glioma cells.CERS1 could induce the activation of ER stress,induction of lethal autophagy and inhibition of PI3K/AKT signal pathway in glioma cells,and cell death induced by overexpression of CERS1 was independent of Bax and Bcl-2.